Methods for amyloid removal using anti-amyloid antibodies

ABSTRACT

Methods and related immunoglobulin peptides and fragments thereof are disclosed that enhance the cell-mediated immune response of a patient to deposits of amyloid fibrils. These methods exploit the opsonizing effect of antibodies directed toward amyloid material or its component parts.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. provisional applicationNo. 60/086,198, filed May 21, 1998, which is herein incorporated byreference in its entirety.

FEDERAL SUPPORT

[0002] This invention was made with government support under Grant No. 2R01 CA 20056, awarded by The National Institutes of Health. Thus, thegovernment may have certain rights in this invention.

TECHNICAL FIELD

[0003] The present invention generally relates to methods for treatingamyloid-related diseases. Specifically, the present invention providestherapeutic antibody-related methods to effect the removal of amyloidfibrils by a patient's own immunophagocytic system.

BACKGROUND OF THE INVENTION

[0004] Amyloidosis refers to the pathological deposition of proteins inthe form of congophilic, green birefringent fibrils, when congored-stained, either dispersed or in the form of localized amyloidomas.Such deposits are symptomatic of several diseases, for exampleAlzheimer's Disease, inflammation-associated amyloid, type II diabetes,bovine spongiform encephalopathy (BSE), Creutzfeld-Jakob disease (CJD),scrapie and primary amyloidosis.

[0005] Amyloidoses are generally categorized into three groups: majorsystemic amyloidoses, major localized amyloidoses, and miscellaneousamyloidoses. Major systemic amyloidoses include: chronic inflammatoryconditions (e.g., tuberculosis, osteomyelitis, etc.); non-infectiousconditions such as juvenile rheumatoid arthritis, ankylosing spondylitisand Crohn's disease, etc.; familial Mediterranean Fever, plasma celldyscrasia (primary amyloidosis) and various familial polyneuropathiesand cardiomyopathies. Major localized amyloidoses include: chronicdialysis usually for greater than 8 years, Alzheimer's disease, Downsyndrome, Hereditary cerebral hemorrhage (Dutch), and non-traumaticcerebral hemorrhage of the elderly. Miscellaneous amyloidoses include:familial polyneuropathy (Iowa), familial amyloidosis (Finnish),hereditary cerebral hemorrhage (Icelandic), CJD, Medullary carcinoma ofthe thyroid, atrial amyloid, and diabetes mellitus (insulinomas). Otheramyloidoses include those referenced in Louis W. Heck, “The AmyloidDiseases” in Cecil's Textbook of Medicine 1504-6 (W. B. Saunders & Co.,Philadelphia, Pa.; 1996).

[0006] Transmissible spongiform encephalopathies which cause CJD andGerstmann-Strässler-Scheinker (GSS) disease are described by B. Chesebroet al., “Transmissible Spongiform Encephalopathies: A BriefIntroduction” in Field's Virology 2845-49 (3rd Edition; RavenPublishers, Philadelphia, Pa.; 1996) and in D. C. Gajdusek, “Infectiousamyloids: Subacute Spongiform Encephalopathies as Transmissible CerebralAmyloidoses,” 2851-2900 in Fields Virology (1996). Many of thesediseases are likely mediated by prions, an infectious protein. See S. B.Prusiner, “Prions” in Fields Virology 2901-50 (1996) and the referencescontained therein. The inherited forms of amyloidoses as described onOnline Mendelian Inheritance in Man (OMIM)“www.ncbi.nlm.nih.gov/htbin-post/Omim/dispmim?” Each of the above isincorporated herein by reference.

[0007] Very rarely do patients with clinically proven amyloidosisspontaneously achieve complete remission, perhaps because the amyloidfibrils themselves are non-immunogenic. Various therapies foramyloidosis have been investigated, such as high-dose chemotherapy,steroids, iodinated doxorubicin, and stem cell replacement therapy.However, in only one type of amyloid disease, Familial-Mediterraneanamyloidosis, has drug treatment (with colchicine) been shown to beeffective.

[0008] The use of monoclonal antibodies (mAbs) to induce or modulate theimmunological removal of an otherwise unrecognized entity is known. mAbshave been successfully used in treating non-Hodgkins lymphoma and breastcancer, for example.

[0009] Previously, a variety of studies have characterized antibodiesthat bind to amyloid proteins or amyloid fibrils. See, for example, U.S.Pat. Nos. 5,714,471; 5,693,478; 5,688,651; 5,652,092; 5,593,846;5,536,640; 5,385,915; 5,348,963; 5,270,165; 5,262,332; 5,262,303;5,164,295; and 4,782,014. In addition, several publications havesuggested that anti-amyloid antibodies might be useful for studying theprogression of beta-amyloidosis and for various therapeutic options.See, for example, Bellottii et al., Scand. J. Immunol. (1992)36(4):607-615; Bellotti et al., Ren. Fail. (1993) 15(3):365-371; Walkeret al. J. Neuropathol. Exp. Neurol. (1994) 53(4):377-383; and Bickel etal., Bioconjug. Chem. (1994) 5(2):119-125. However, no therapeuticantibody has been demonstrated to halt or reverse the deposition ofamyloid fibrils in a patient. Thus, a need exists for a method fortreating amyloidoses using antibody formulations containing antibodiesthat bind to amyloid fibrils.

SUMMARY OF THE INVENTION

[0010] The present inventors have discovered new methods of treatingamyloid-related diseases and conditions. These methods exploit theopsonizing effect of mAbs directed toward the protein constituents ofamyloid.

[0011] The present invention includes a method of treating a patienthaving an amyloid-associated disease comprising the step ofadministering to the patient a therapeutically effective dose of atleast one immunoglobulin polypeptide, or fragments thereof, togetherwith a pharmaceutically acceptable carrier; wherein the immunoglobulinpolypeptide or fragment thereof, may be a substantially purifiedimmunoglobulin polypeptide that binds to a human amyloid fibril, whereinbinding of the polypeptide opsonizes the amyloid fibril.

[0012] In particular, the present invention relates to the use of anyone of, or a combination of, the three monoclonal antibodies discussedbelow. These antibodies have general anti-amyloid binding properties andprovide an extrinsic opsonizing reagent that activates a patient's owncellular immune clearance mechanism.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013]FIGS. 1A and 1B. FIGS. 1A and 1B are reproduced photographs of aBalb/c mouse just after an injection of amyloid is made (1A) and 14 daysafter the injection (1B). The injection site was shaved to betterillustrate the “hump” caused by the injection of the amyloid material.

[0014] FIGS. 2A-2B. FIGS. 2A and 2B are reproduced photographs of humanneutrophils (multi-lobed nuclei) adhering to human amyloid opsonized invitro.

[0015] FIGS. 3A-3D. FIGS. 3A-3D are reproduced photographs ofimmunohistochemically stained amyloid-laden tissue samples (20Xmagnification). FIG. 3A is a tissue sample from a patient with κ1amyloidosis stained with Congo red; the amyloid deposits, viewed underpolarized light, appear as blue-green particles. FIG. 3B is a tissuesample stained with alkaline phosphatase after labeling with anti-κI(57-18-H12) mAb. FIG. 3C is a tissue sample stained as in FIG. 3B, butwith anti-κIV (11-1F4) mAb. FIG. 3D is a tissue sample stained as inFIG. 3B, but with anti-λVIII (31-8c7) mAb.

[0016]FIG. 4. FIG. 4 is a reproduced photograph showing afluoresceinated (FITC) κ4 mAb bound to human amyloid implanted into aBalb/c mouse. The mAb was injected into the thigh of the mouse. Theamyloidoma was excised 72 hours post injection and viewed using anepifluorescence microscope (20X magnification).

MODES OF CARRYING OUT THE INVENTION

[0017] General Description

[0018] The present invention utilizes immunoglobulin polypeptides tomodulate and to enhance the degradation and removal of undesireddeposits of amyloid fibrils in a host or patient. It is envisioned thatthe invention will be used, for example, to treat humans suffering froma disease or condition characterized by an undesired deposition ofamyloid fibrils. Without intending to be bound by any particularmechanism of action, it is believed that the administration ofimmunoglobulin peptides according to the present invention opsonize thedeposited amyloid fibrils in a patient suffering from amyloidosis,thereby assisting in their removal from the patient by the patients' ownimmune system. It is believed that the patient's immune system alone isunable to remove the amyloid fibrils in conditions modulated by amyloidfibrils without such a therapeutic intervention, presumably because theamyloid fibrils are themselves relatively non-immunogenic.

[0019] To treat a patient with amyloidosis, a therapeutically effectivedose of immunoglobulin polypeptide or fragment thereof according to thepresent invention is administered together with a pharmaceuticallysuitable carrier or excipient. Upon the binding or adhering of suchimmunoglobulin polypeptides to undesired deposits of amyloid fibrils,the latter are believed to be opsonized.

[0020] Single or multiple administrations of the compositions of thepresent invention can be carried out in dosages and by administrationprotocols known to those skilled in the art for the administration ofother therapeutic antibody products. These parameters may be selectedand/or optimized by the physician treating a particular patient.

[0021] Preferably, a therapeutically effective dose of a pharmaceuticalformulation of the present invention should deliver a quantity ofanti-amyloid immunoglobulin polypeptide sufficient to substantiallyinhibit the undesired deposition of amyloid fibrils or to substantiallyinhibit the rate of any undesired deposition of amyloid fibrils. Morepreferably, the formulations should reduce the overall burden ofdeposited amyloid fibrils in a patient. Further, administration of suchformulations should begin shortly after diagnosis of amyloidosis andcontinue until symptoms are substantially abated and for a periodthereafter. In well established cases of disease, loading doses followedby maintenance doses may be required.

[0022] Definitions

[0023] The terms “peptide,” “polypeptide” or “protein” are usedinterchangeably herein. The term “substantial identity,” when referringto polypeptides, indicates that the polypeptide or protein in questionis at least about 30% identical to an entire naturally occurring proteinor a portion thereof, usually at least about 70% identical, andpreferably at least about 95% identical.

[0024] As used herein, the terms “isolated,” “substantially pure” and“substantially homogenous” are used interchangeably and describe aprotein that has been separated from components which naturallyaccompany it. A substantially purified protein will typically compriseover about 85% to 90% of a protein sample, more usually about 95%, andpreferably will be over about 99% pure. Protein purity or homogeneitymay be indicated by a number of means well known in the art, such apolyacrylamide gel electrophoresis of a protein sample, followed byvisualizing a single polypeptide band on a polyacrylamide gel uponstaining. For certain purposes high resolution will be needed and HPLCor a similar means for purification utilized.

[0025] Proteins may be purified to substantial homogeneity by standardtechniques well known in the art, including selective precipitation withsuch substances as ammonium sulfate, column chromatography,immunopurification methods, and others. See, for instance, Scopes,Protein Purification: Principles and Practice, Springer-Verlag: New York(1982), which is incorporated herein by reference.

[0026] Antibody purification techniques are well known in the art.Harlow et al., Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor (1988), 288-318, which isincorporated herein by reference, describes, for example, purificationusing ammonium sulfate precipitation, caprlic acid, DEAE, hydroxyapatitechromatography, gel filtration chromatography, protein A beads, andimmunoaffinity.

[0027] Nucleic acids, as used herein, may be DNA or RNA. When referringto nucleic acids, the term “substantial identity” indicates that thesequences of two nucleic acids, or designated portions thereof, whenoptimally aligned and compared, are identical, with appropriatenucleotide insertions or deletions, in at least about 80% of thenucleotides, usually at least about 90% to 95%, and more preferably atleast about 98% to 99.5% of the nucleotides.

[0028] Alternatively, substantial nucleic acid sequence identity existswhen a nucleic acid segment will hybridize under selective hybridizationconditions, to a complement of another nucleic acid strand.

[0029] “Substantially complementary” similarly means that one nucleicacid hybridizes selectively to, or is identical to, another nucleicacid. Typically, selective hybridization will occur when there is atleast about 55% identity over a stretch of at least 14-25 nucleotides,preferably at least about 65% identity, more preferably at least about75%, and most preferably at least about 90% identity. See M. KanehisaNucleic Acids Res. 12:203 (1984), which is incorporated herein byreference.

[0030] Stringent hybridization conditions will typically include saltconcentrations of less than about 1 M, more usually less than about 500mM and preferably less than about 200 mM. Temperature conditions willtypically be greater than 22° C., typically greater than about 30° C.and preferably in excess of about 37° C. As other factors maydramatically affect the stringency of hybridization, including basecomposition and size of the complementary strands, presence of organicsolvents and extent of base mismatching, the combination of parametersis more important than the absolute measure of any one alone.

[0031] “Isolated” or “substantially pure,” when referring to nucleicacids, refer to those that have been purified away from other cellularcomponents or other contaminants, e.g., other cellular nucleic acids orproteins, by standard techniques, including alkaline/SDS treatment, CsClbanding, column chromatography, and others well known in the art. See,F. Ausubel, et al., ed. Current Protocols in Molecular Biology, GreenePublishing and Wiley-Interscience, New York (1987), incorporated hereinby reference.

[0032] A nucleic acid is “operably linked” when it is placed into afunctional relationship with another nucleic acid sequence. Forinstance, a promoter or enhancer is operably linked to a coding sequenceif it affects the transcription of the sequence. Generally, operablylinked means that the nucleic acid sequences being linked are contiguousand, where necessary to join two protein coding regions, contiguous andin reading frame.

[0033] Techniques for nucleic acid manipulation, such as subcloningnucleic acid sequences encoding polypeptides into expression vectors,labelling probes, DNA hybridization, and so on are described generally,for example in Sambrook et al., (1989) Molecular Cloning: A LaboratoryManual (2nd ed.), Vols. 1-3, Cold Spring Harbor Laboratory, or Ausubelet al., ed. (1987) op. cit., both of which are incorporated herein byreference.

[0034] “Expression vectors,” “cloning vectors,” or “vectors” are oftenplasmids or other nucleic acid molecules that are able to replicate in achosen host cell. Expression vectors may replicate autonomously, or theymay replicate by being inserted into a genome of the host cell, bymethods well known in the art. Vectors that replicate autonomously willhave an origin of replication or autonomous replicating sequence (“ARS”)that is functional in the chosen host cell(s). Often, it is desirablefor a vector to be usable in more than one host cell, e.g., in E. colifor cloning and construction, and in a mammalian cell for expression.

[0035] Mammalian cell lines are often used as host cells for theexpression of polypeptides derived from eukaryotes. Propagation ofmammalian cells in culture is per se well known. See, Tissue Culture,Academic Press, Kruse and Patterson, ed. (1973), incorporated herein byreference. Host cell lines may also include such organisms as bacteria(e.g., E. coli or B. subtilis), yeast, filamentous fungi, plant cells,or insect cells, among others.

[0036] “Transformation” refers to the introduction of vectors containingthe nucleic acids of interest directly into host cells by well-knownmethods. Transformation methods, which vary depending on the type ofhost cell, include electroporation; transfection employing calciumchloride, rubidium chloride calcium phosphate, DEAE-dextran, or othersubstances; microprojectile bombardment; lipofection; infection (wherethe vector is an infectious agent); and other methods. See generally,Sambrook et al., (1989) op. cit. Reference to cells into which thenucleic acids described above have been introduced is meant to alsoinclude the progeny of such cells.

[0037] As used herein, “immunoglobulin polypeptide” refers to moleculesthat are derived from native immunoglobulins (e.g., antibodies) thathave specific immunoreactive activity against a particular target, e.g.,against amyloid fibrils. Antibodies are typically tetramers ofimmunoglobulin polypeptides. As used herein, the term “antibody” alsorefers to a protein consisting of one or more polypeptides substantiallyencoded by immunoglobulin genes. Immunoglobulin genes include thosecoding for the light chains, which may be of the kappa or lambda types,and those coding for the heavy chains. Heavy chain types are alpha,gamma, delta, epsilon and mu. The carboxy terminal portions ofimmunoglobulin heavy and light chains are constant regions, while theamino terminal portions are encoded by the myriad immunoglobulinvariable region genes. The variable regions of an immunoglobulin are theportions that provide antigen recognition specificity. In particular,the specificity resides in the complementarity determining regions(“CDRs”), also known as hypervariable regions, of the immunoglobulins.

[0038] The immunoglobulins may exist in a variety of fragment formsincluding, for example, Fv, Fab, F(ab″), F(ab′)₂, SvFv and otherfragments, as well as single chains (e.g., Huston, et al., Proc. Nat.Acad. Sci. U.S.A., 85:5879-5883 (1988) and Bird, et al., Science242:423-426 (1988), which are incorporated herein by reference). (See,generally, Hood, et al., “Immunology,” Benjamin, N.Y., 2nd ed. (1984),and Hunkapiller and Hood, Nature, 323:15-16 (1986), which areincorporated herein by reference). Single-chain antibodies, in whichgenes for a heavy chain and a light chain are combined into a singlecoding sequence, may also be used. Immunoglobulin polypeptide alsoencompasses a truncated immunoglobulin chain, for example, a chaincontaining less constant region domains than in the native polypeptide.Such truncated polypeptides can be produced by standard methods such asintroducing a stop codon into the gene sequence 5′ of the domainsequences to be deleted. The truncated polypeptides can then beassembled into truncated antibodies. Antibodies as used herein alsoinclude bispecific antibodies which can be produced such as by themethods described in the following references: Glennie et al., J.Immunol., 139:2367-2375 (1987); Segal et al., Biologic Therapy of CancerTherapy of Cancer Updates 2(4):1-12 (1992); and Shalaby et al., J. Exp.Med. 175:217-225 (1992).

[0039] “Monoclonal antibodies” may be obtained by various techniquesfamiliar to those skilled in the art. Briefly, spleen cells from ananimal immunized with a desired antigen are immortalized, commonly byfusion with a myeloma cell (see Kohler and Milstein, Eur. J. Immunol.6:511-519 (1976)). Alternative methods of immortalization includetransformation with Epstein Barr Virus, oncogenes, or retroviruses, orother methods well known in the art. Colonies arising from singleimmortalized cells are screened for production of antibodies of thedesired specificity and affinity for the antigen, and yield of themonoclonal antibodies produced by such cells may be enhanced by varioustechniques, including injection into the peritoneal cavity of avertebrate host.

[0040] Monospecific and bispecific immunoglobulins may also be producedby recombinant techniques in prokaryotic or eukaryotic host cells.

[0041] “Chimeric” antibodies are encoded by immunoglobulin genes thathave been genetically engineered so that the light and heavy chain genesare composed of immunoglobulin gene segments belonging to differentspecies. For example, the variable (V) segments of the genes from amouse monoclonal antibody may be joined to human constant (C) segments.Such a chimeric antibody is likely to be less antigenic to a human thanantibodies with mouse constant regions as well as mouse variableregions.

[0042] As used herein, the term chimeric antibody also refers to anantibody that includes an immunoglobulin that has a human-like frameworkand in which any constant region present has at least about 85%-90%, andpreferably about 95% polypeptide sequence identity to a humanimmunoglobulin constant region, a so-called “humanized” immunoglobulin(see, for example, PCT Publication WO 90/07861, which is incorporatedherein by reference). Hence, all parts of such a “humanized”immunoglobulin, except possibly the complementarity determining regions(CDRs), are substantially identical to corresponding parts of one ormore native human immunoglobulin sequences. Where necessary, frameworkresidues may also be replaced with those within or across speciesespecially if certain framework residues are found to affect thestructure of the CDRs. A chimeric antibody may also contain truncatedvariable or constant regions.

[0043] The term “framework region,” as used herein, refers to thoseportions of immunoglobulin light and heavy chain variable regions thatare relatively conserved (i.e., other than the CDRs) among differentimmunoglobulins in a single species, as defined by Kabat, et al.,(1987); Sequences of Proteins of Immunologic Interest, 4th Ed., U.S.Dept. Health and Human Services, which is incorporated herein byreference). As used herein, a “human-like framework region” is aframework region that in each existing chain comprises at least about 70or more amino acid residues, typically 75 to 85 or more residues,identical to those in a human immunoglobulin.

[0044] Human constant region DNA sequences can be isolated in accordancewith well-known procedures from a variety of human cells, but preferablyfrom immortalized B-cells. The variable regions or CDRs for producingthe chimeric immunoglobulins of the present invention may be similarlyderived from monoclonal antibodies capable of binding to the human typeamyloid, and will be produced in any convenient mammalian system,including mice, rats, rabbits, human cell lines, or other vertebratescapable of producing antibodies by well-known methods. Variable regionsor CDRs may be produced synthetically, by standard recombinant methods,including polymerase chain reaction (“PER”) or through phage-displaylibraries. For phage display methods, see for example, McCafferty etal., Nature 348:552-554 (1990); Clackson et al., Nature 352:624-628 andMarks et al., Biotechnology 11:1145-1149 (1993). Suitable prokaryoticsystems such as bacteria, yeast and phage may be employed.

[0045] Suitable source cells for the DNA sequences and host cells forimmunoglobulin expression and secretion can be obtained from a number ofsources, such as the American Type Culture Collection (“Catalogue ofCell Lines and Hybridomas,” Fifth edition (1985) Rockville, Md., U.S.A.,which is incorporated herein by reference).

[0046] In addition to the chimeric and “humanized” immunoglobulinsspecifically described herein, other substantially identical modifiedimmunoglobulins can be readily designed and manufactured utilizingvarious recombinant DNA techniques well known to those skilled in theart. In general, modifications of the genes may be readily accomplishedby a variety of well-known techniques, such as PCR and site-directedmutagenesis (see, Gillman and Smith, Gene 8:81-97 (1979) and S. Robertset al., Nature 328:731-734 (1987), both of which are incorporated hereinby reference).

[0047] Alternatively, polypeptide fragments comprising only a portion ofthe primary immunoglobulin structure may be produced. For example, itmay be desirable to produce immunoglobulin polypeptide fragments thatpossess one or more immunoglobulin activities in addition to, or otherthan, antigen recognition (e.g., complement fixation).

[0048] Immunoglobulin genes, in whole or in part, may also be combinedwith functional regions from other genes (e.g., enzymes), or with othermolecules such as toxins, labels and targeting moieties to producefusion proteins (e.g., “immunotoxins”) having novel properties. In thesecases of gene fusion, the two components are present within the samepolypeptide chain. Alternatively, the immunoglobulin or fragment thereofmay be chemically bonded to the toxin or label by any of a variety ofwell-known chemical procedures. For example, when the label or cytotoxicagent is a protein and the second component is an intact immunoglobulin,the linkage may be by way of heterobifunctional cross-linkers, e.g.,SPDP, carbodiimide, glutaraldehyde, or the like.

[0049] Suitable labels include, for example, radionuclides, enzymes,substrates, cofactors, inhibitors, fluorescers, chemiluminescers,magnetic particles. See, for examples of patents teaching the use ofsuch labels, U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345;4,277,437; 4,275,149; and 4,366,241, all of which are incorporated byreference.

[0050] Immunotoxins, including single chain molecules, may also beproduced by recombinant means. Production of various immunotoxins iswell-known with the art, and methods can be found, for example in“Monoclonal Antibody-Toxin Conjugates: Aiming the Magic Bullet,” Thorpeet al., Monoclonal Antibodies in Clinical Medicine, Academic Press, pp.168-190 (1982); E. Vitetta, Science (1987) 238:1098-1104; and G. Winterand C. Milstein, Nature (1991) 349:293-299; all incorporated herein byreference.

[0051] Additional techniques for preparing immunoglobulins andimmunoglobulin fragments are described in V. S. Malik et al, AntibodyTechniques (Academic Press, 1994); C. A. K. Borrebaeck, AntibodyEngineering: Breakthroughs in Molecular Biology (Oxford Univ. Press,1995); and P. J. Delves et al., Antibody Production: EssentialTechniques (John Wiley & Sons, 1997), which are incorporated herein byreference.

[0052] “Opsonize”, as used herein, refers to the binding of animmunoglobulin polypeptide to a particular target, particularly epitopesfound on deposits of amyloid fibrils, such that the antibody and targetstogether are recognized as “foreign” by the host's cellular immunesystem. In other words the binding of the immunoglobulin of the presentinvention enhances the phagocytization of the amyloid fibrils.

[0053] “Amyloidosis”, as used herein, is intended to refer to anycondition that is characterized by the presence of amyloid material.Such material may be in the form of an amyloidoma or more disperseamyloid deposits or fibrils.

[0054] Pharmaceutical Compositions

[0055] The pharmaceutical compositions for therapeutic treatmentaccording to the present invention are intended for parenteral, oral orlocal administration. Preferably, the pharmaceutical compositions areadministered parenterally, e.g., intravenously, subcutaneously,intradermally, or intramuscularly. As the blood brain barrier isimpermeable to IgG (see U. Bickel et al., 1994 Bioconjug. Chem. 5:119-25), delivery of antibodies to overcome the blood-brain barrier(BBB) may be achieved through liposomal or micellar delivery of theantibody to the desired site. Alternatively, the agents of thisinvention can be delivered directly into the cerebrospinal fluid (seefor example L. C. Walker et al., 1994 J. Neuropathol. Exp. Neurol. 53:377-83). For other delivery mechanisms, refer to P. M. Friden, 1996 U.S.Pat. No. 5,527,527 and W. M. Pardridge, 1991 U.S. Pat. No. 5,004,697.All of the above documents are incorporated herein by reference.

[0056] Thus, the invention provides compositions for parenteraladministration which comprise a solution of the anti-amyloidimmunoglobulin polypeptide dissolved or suspended in a pharmaceuticallyacceptable carrier, preferably an aqueous carrier. A variety of aqueouscarriers may be used, e.g., water, buffered water, 0.4% saline, 0.3%glycine, hyaluronic acid and the like. These compositions may besterilized by conventional, well known sterilization techniques, or maybe sterile filtered. The resulting aqueous solutions may be packaged foruse as is, or lyophilized, the lyophilized preparation being combinedwith a sterile solution prior to administration. The compositions maycontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents, wetting agents and the like, forexample, sodium acetate, sodium lactate, sodium chloride, potassiumchloride, calcium chloride, sorbitan monolaurate, triethanolamineoleate, etc.

[0057] The concentration of anti-amyloid immunoglobulin polypeptides ofthe invention in the pharmaceutical formulations can vary widely, i.e.,from less than about 1%, usually at or at least about 10-15% to as muchas 50% or more by weight, and will be selected primarily by fluidvolumes, viscosities, etc., in an accordance with the particular mode ofadministration selected.

[0058] Without undue experimentation, one of ordinary skill in the artcould determine the quantity of immunoglobulin polypeptides that wouldbe effective in adequately opsonizing an amyloidoma. Amounts effectivefor this use will depend on, e.g., the nature of the anti-amyloidimmunoglobulin polypeptide composition, the manner of administration,the stage and severity of the disease being treated, the weight andgeneral state of health of the patient, and the judgment of theprescribing physician. A typical single dose of 0.5 mg/kg couldgenerally be used. It must be kept in an mind that the anti-amyloidimmunoglobulin polypeptide and peptide compositions derived therefrommay be employed in serious disease states, that is, life-threatening orpotentially life-threatening situations. In such cases it is possibleand may be felt desirable by the treating physician to administersubstantial excesses of these compositions. Thus, human anti-amyloidmonoclonal antibodies or substantially human anti-amyloid receptormonoclonal antibodies of the invention are most preferred under thesecircumstances.

[0059] Treatment of humans with amyloidosis according to the presentinvention could also be applied to animals susceptible to amyloidosis,such as cows or chickens. Thus, references to human patients hereinapply also to non-human patients.

[0060] The immunoglobulin polypeptides, as defined herein, arepreferably anti-amyloid mAbs directed toward an amyloidoma or componentsor precursors thereof. The mAbs can be raised against IgLC variableregion domains or, preferably, against the IgLC subsets κ1, κ4, λ8, orcombinations thereof. The administration to humans of immunoglobulinpolypeptides that are substantially non-human may elicit anti-antibodyresponses. Thus, it may be desirable to prepare anti-IgLC immunoglobulinpolypeptides of the present invention which are substantially human. By“substantially human” is meant an antibody or binding fragment thereofcomprised of amino acid sequences which are at least about 50% human inorigin, at least 70 to 80% more preferred, and about 95-99% or morehuman most preferred, particularly for repeated administrations over aprolonged period as may be necessary to treat established cases ofamyloidosis. As used herein, human antibody is meant to includeantibodies of entirely human origin as well as those which aresubstantially human, unless the context indicates otherwise.

[0061] Monoclonal antibodies can also be raised against syntheticamyloid fibrils. Recombinant light chain, variable region peptides areisolated and purified in vitro using standard techniques. Syntheticfibrils are then prepared from the peptides using techniques such asthose described by Wall et al., “In vitro Immunoglobulin Light ChainFibrillogenesis,” METHODS IN ENZYMOLOGY, Vol. 309 (In Press). Antibodiesare then raised against the synthetic fibrils using standardimmunization techniques, typically in mice or rabbits. Monoclonal celllines secreting anit-fibril antibodies are produced using standardhybridoma techniques.

[0062] The anti-amyloid immunoglobulin polypeptides of the invention maybe prepared by any of a number of well-known techniques. For instance,they may be prepared by immunizing an animal with purified or partiallypurified human amyloid. The animals immunized can be any one of avariety of species which are capable of immunologically recognizingepitopes characteristic of the human type amyloid extracellular domain,such as murine, lagomorph, equine, etc.

[0063] Monoclonal antibodies of the invention may be prepared byimmortalizing cells comprising nucleic acid sequences which encodeimmunoglobulin polypeptides or portions thereof that bind specificallyto antigenic determinants characteristic of the extracellular domain ofthe human type amyloid. The immortalization process can be carried outby hybridoma fusion techniques, by viral transformation ofantibody-producing lymphocytes, recombinant DNA techniques, or bytechniques that combine cell fusion, viral transformation and/orrecombinant DNA methodologies. Immunogens to raise the monoclonalantibodies include synthetic amyloid fibrils as described, for exampleby, A. Lomakin et al., 1997 Proc. Nat'l Acad. Sci. USA 94: 7942-7, whichis incorporated herein by reference.

[0064] As the generation of human anti-amyloid monoclonal antibodies maybe difficult with conventional immortalization techniques, it may bedesirable to first make non-human antibodies and then transfer viarecombinant DNA techniques the antigen binding regions of the non-humanantibodies, e.g., the Fab, complementarity determining regions (CDRs) orhypervariable regions, to human constant regions (Fc) or frameworkregions as appropriate to produce substantially human molecules. Suchmethods are generally known in the art and are described in, forexample, U.S. Pat. No. 4,816,397, PCT publication WO 90/07861, and EPpublications 173494 and 239400, wherein each is incorporated herein byreference. However, completely human antibodies can be produced intransgenic animals. The desired human immunoglobulin genes or genesegments can be isolated, for example by PCR from human B cells, the DNAcloned into appropriate vectors for expression in eukaryotic cells andthe cloned DNA introduced into animals to produce transgenics. Animalssuitable for the production of transgenics expressing humanimmunoglobulin include mice, rats, rabbits and pigs with rodents oftransgenics that express human immunoglobulins should preferably haveone or more of their endogenous immunoglobulin loci inactivated or“knocked-out” to facilitate identification and isolation of the humanantibodies (See e.g., Lonberg, et al. Nature 368:856-859 (1994)).

[0065] The resulting chimeric antibodies or chimeric immunoglobulinpolypeptides that bind to human amyloid are also within the scope of thepresent invention. A typical therapeutic chimeric antibody would be ahybrid protein consisting of the variable (V) or antigen-binding domainfrom a mouse immunoglobulin specific for a human amyloid antigenicdeterminant, and the constant (C) or effector domain from a humanimmunoglobulin, although domains from other mammalian species may beused for both variable and constant domains. As used herein, the therm“chimeric antibody” also refers to antibodies coded for byimmunoglobulin genes in which only the CDRs are transferred from theimmunoglobulin that specifically recognizes the antigenic determinants,the remainder of the immunoglobulin gene being derived from a human (orother mammalian, as desired) immunoglobulin gene. As discussed before,this type of chimeric antibody is referred to as a “humanized” (in thecase of a human immunoglobulin gene being used) antibody. Alsoconsidered are recombinant human antibodies that do not containsequences of another species.

[0066] The hypervariable regions of the variable domains of theanti-amyloid immunoglobulin polypeptides comprise a related aspect ofthe invention. The hypervariable regions, or CDRs, in conjunction withthe framework regions (those portions of immunoglobulin light and heavychain variable regions that are relatively conserved among differentimmunoglobulins in a single species), enable the anti-amyloidimmunoglobulin polypeptides to recognize and thus bind to human amyloid.The hypervariable regions can be cloned and sequenced. Once identified,these regions that confer specific recognition of human amyloid can thenbe cloned into a vector for expression in a host as part of anotherimmunoglobulin molecule or as a fusion protein, e.g., a carrier moleculewhich functions to enhance immunogenicity of the cloned idiotype.

[0067] The anti-amyloid immunoglobulin polypeptides of the inventionwill generally be used intact, or as immunogenic fragments, such Fv,Fab, F(ab′)₂ fragments. The fragments may be obtained from antibodies byconventional techniques, such as by proteolytic digestion of theantibody using, e.g., pepsin, papain or other proteolytic enzymes, or byrecombinant DNA techniques in which a gene or portion thereof encodingthe desired fragment is cloned or synthesized, and expressed in avariety of hosts.

[0068] Those skilled in the art will realize that “anti-idiotypic”antibodies can be produced by using a specific immunoglobulin as animmunogen in accordance with standard techniques. For example, infectionor immunization with an amyloid fibril or fragment thereof, induces aneutralizing immunoglobulin, which has on its Fab variable regioncombining site an image of the amyloid that is unique to that particularimmunoglobulin, i.e., an idiotype. Immunization with such ananti-amyloid immunoglobulin induces an anti-idiotype antibody, which hasa conformation at its combining site that mimics the structure of theoriginal amyloid antigen. These anti-idiotype antibodies may thereforebe used instead of the amyloid antigen. See, for example, Nisonoff(1991) J. Immunol. 147:2429-2438, which is incorporated herein byreference.

[0069] The following working examples specifically point out preferredembodiments of the present invention, and are not to be construed aslimiting in any way the remainder of the disclosure. Other genericconfigurations will be apparent to one skilled in the art.

EXAMPLE 1 Unassisted Resolution of Human IgLC Amyloid in Murine Host

[0070] Human IgLC amyloid was extracted and purified from infectedorgans obtained during an autopsy. The first experiments involvedtransplanting 50-200 mg of this amyloid material into a Balb/c mouse.The amyloid mass, or “amyloidoma,” was prepared in sterile PBS by serialsonication and grinding steps in order to produce a fine suspension ofamyloid fibrils complete with the accessory molecules found in vivo.This procedure was performed to allow the amyloid to be injected intothe mice through a wide-gauge hypodermic needle.

[0071] The amyloid material, equivalent to 10% of the body weight of theanimal, was injected into mice (under anesthetic) between the scapula,which resulted in a large mass being visible (see FIG. 1A). The mouserequired 15-18 days to achieve the complete removal of the amyloidoma(see FIG. 1B), after which the animal appeared healthy and lived anormal life span. The removal of the amyloidoma was determinedsubjectively by the experimenter; by simply palpating the injectionsite, an amyloidoma, like a hard pea, can be easily felt under the skin.

EXAMPLE 2 Involvement of Both Antibody-Meditated and Cellular Immunityin the Removal of Amyloidomas

[0072] The involvement of anti-amyloid antibodies in the removal ofamyloidomas was shown by screening serum from a mouse previouslyinjected with amyloid material against a sample of the injectedmaterial. This was done by Western blot analysis using suitabledilutions of the mouse serum as the primary antibody. It was shown thatthere were antibodies to every component of the amyloid matrix, i.e.,every band on the gel was stained by the mouse serum, even at a10,000-fold serum dilution (data not shown).

[0073] The involvement of a cellular component was demonstrated by invitro neutrophil binding assays (see FIGS. 2A and 2B) and by usingknockout-mutant mouse strains (data not shown). FIGS. 2A and 2B showhuman neutrophils adhering to human amyloid after the amyloid wastreated with mouse anti-human IgLC mAbs. This shows that the mouse mAbcan bind to human amyloid as well as attract human neutrophils.

[0074] Studies of knockout-mutant mouse strains further support afinding of antibody involvement in amyloid removal. First, scid/scidmice, which lack B and T lymphocytes, were unable to remove an injectedamyloidoma even after three months (data not shown). Second, CD18knockout animals were unable to remove the amyloidoma as rapidly asnormal animals. CD18 knockout animals are 97% deficient in CD18, a cellsurface integrin found on granulocyte/macrophage lineages. Althoughthese cells cannot leave the circulation, the animals are B and T cellcompetent and can therefore mount an antibody response. Third, nudemice, which have no white blood cells, were unable to remove theamyloidoma.

[0075] Furthermore, amyloid that had been incubated withamyloid-reactive serum from another mouse, when implanted into thesecond mouse, was removed within 4 days. In this experiment a Balb/cmouse was injected with 50 mg HIG amyloid and left for 1 week, afterwhich it was bled by tail-vein clipping. The blood was spun down at 1500rpm and the cells removed by aspiration. The plasma was stored at 4° C.until used. Another preparation of HIG amyloid (100 mg) was prepared bysuspending in sterile PBS to which was added 1 ml of plasma from theprevious mouse. This preparation was then injected into a second mouse(Balb/c) and the amyloid was removed in 4 days. Thus, it was concludedthat the process could be sped up by opsonizing the material prior toinjection.

EXAMPLE 3 ELISA Screening of IgLC Subsets

[0076] A systematic study was performed using ELISA techniques to screena large number of human extracted amyloid samples using mAbs raisedagainst the IgLC subsets (λ1, λ2, λ3, λ4, λ5, λ6, κ1, κ2, κ3, κ4, free κand λ and total κ and λ). Interestingly, it was found that more oftenthan not, the amyloids tested positive with mAbs specific for their ownsubtype, the total κ or λ antibodies and a κ1(57-18H12), κ4(11-1F4) andλ8(31-8C7) mAb. These latter three reagents were found to react in anon-subgroup specific manner, i.e., κ1 reacted with amyloids comprisedof IgLCs other than κ1; and the other two mAbs exhibit the same quality.This shows that the epitope recognized by these antibodies may be ageneral feature of amyloid fibrils, indicating the possibility of ashared amyloid epitope that can be targeted.

EXAMPLE 4 Immunochemical Staining

[0077] Tissue samples from amyloid patients were stained using standardimmunochemical techniques and a similar binding phenomenon was observed.FIGS. 3A-3D show that anti-κ1 binds to the κ1 amyloid and, surprisingly,that the anti-κ4 reacts with the κ1 amyloid, suggesting an amyloidepitope that these antibodies may recognize. Additionally, the anti-κ4reacts with λ-containing amyloid (not illustrated). This is an exampleof cross-isotype reactivity. However, the results from the ELISA and theimmunohistochemistry were not always consistent. This is likely due tothe inherent difference in what you are looking at, i.e., ELISA is aliquid phase binding assay using extracted purified amyloid, whereasimmunohistochemistry is performed on fixed tissue sections on a slide.

[0078] Samples of hybridoma cells that secrete anti-κ1 (57-18-H12 (ATCCAcc. No. ______)), anti-κ4 (11-1F4 (ATCC Acc. No. ______)) and anti-κ8(31-8c7 (ATCC Acc. No. ______)) monoclonal antibodies were depositedwith the American Type Culture Collection (ATCC) on May 21, 1999 incompliance with the Budapest Treaty.

EXAMPLE 5 In Vivo Studies of Anti-IgLC Subgroups

[0079] 0.1 mg of one of three antibodies—κ1, κ4, or λ8, identifiedabove—was injected into the thigh of a mouse into which amyloid had beenintroduced in the form of an amyloidoma as described above. The κ1 andκ4 reagents resulted in the complete removal by the host of most amyloidfibril species tested within 7 days (as little as 4 days for certainsources of amyloid). FIG. 4 shows fluoresceinated κ4 mAb binding tohuman amyloid.

[0080] The λ8 reagent, which is reactive in certain instances in both invitro studies (above), increased the resolution of amyloidomas by up toabout 10% in in vivo experiments.

EXAMPLE 6 In Vivo Studies of Anti-IgLC Subgroups

[0081] Human amyloid was isolated from a patient withinflammation-associated, AA-amyloid and prepared for injection intoBalb/C mice by repeated sonication and grinding in order to permit itsinjection into the mouse (see Example 1). Immediately after theinjection of 100 mg of human AA-amyloid extract, the mice were treatedwith 100 μg of κ4 mAb, anti-AA mAb, no mAb and non-specific control mAb(anti-free κ). Complete resolution of the material was observed with 48hours in the animals that had been treated with the κ4 and anti-AA mAbs.In contrast, the control animals had a large mass of amyloid remainingat the site of injection.

EXAMPLE 7 Production of Specific Anti-Amyloid Fibril mAbs

[0082] Synthetic amyloid fibrils were prepared in vitro and used as animmunogen in mice to produce a first generation of anti-amyloid fibrilmAbs. Briefly, recombinant λ6-light chain, variable region peptides wereproduced, isolated and purified using a bacterial expression system andstandard protein purification techniques. Synthetic fibrils wereprepared from these peptides by extended periods of agitation insolution as described, for example, in Wall et al., “In vitroImmunoglobulin Light Chain Fibrillogenesis,” METHODS IN ENZYMOLOGY, Vol.309 (In Press), which is incorporated herein by reference in itsentirety. Fibrils were concentrated by centrifugation at 17,000×g for 20minutes at room temperature.

[0083] The concentrated fibrils were then used to immunize Balb/c miceover a period of several weeks. Monoclonal cell lines secretinganti-fibril antibodies were produced using standard hybridomatechniques. The resultant antibodies have demonstrable anti-fibrilactivity based upon ELISA assays, described in Example 3. Theseantibodies reacted with 99% of all human IgLC amyloid extracts tested todate irrespective of the nature of the isotype or subgroup of theprecursor protein when tested by ELISA. Similarly, the antibodiesreacted in an ELISA format with isolated murine AA-amyloid and syntheticfibrils composed of a peptide derived from the Alzheimer's protein Aβ[Aβ(25-35)].

[0084] It should be understood that the foregoing discussion andexamples merely present a detailed description of certain preferredembodiments. It therefore should be apparent to those of ordinary skillin the art that various modifications and equivalents can be madewithout departing from the spirit and scope of the invention. Allreferences, articles and patents identified above are hereinincorporated by reference in their entirety.

What is claimed is:
 1. A method of treating a patient having an amyloiddeposition disease comprising the step of administering to the patienta) a therapeutically effective dose of at least one immunoglobulinpolypeptide or a fragments thereof, wherein the immunoglobulinpolypeptide or fragment thereof binds to an amyloid fibril; and b) apharmaceutically acceptable carrier.
 2. The method of claim 1, whereinthe immunoglobulin polypeptide or fragment thereof is raised against animmunoglobulin light-chain.
 3. The method of claim 1, wherein binding ofthe immunoglobulin polypeptide or fragment thereof opsonizes the amyloidfibril.
 4. The method of claim 1, wherein the immunoglobulin polypeptideor fragment thereof is a monoclonal antibody.
 5. The method of claim 4,wherein the monoclonal antibody is a humanized antibody.
 6. The methodof claim 4, wherein the monoclonal antibody is a chimeric antibody. 7.The method of claim 6, wherein the chimeric antibody is a humanizedantibody.
 8. The method of claim 4, wherein the antibody is a labeledantibody.
 9. The method of claim 4, wherein the monoclonal antibody isselected from the group consisting of κ1 (57-18H12), κ4 (11-1F4), λ8(31-8C7), and combinations thereof.
 10. An immunoglobulin polypeptide orfragment thereof that binds to an amyloid fibril and is effective toenhance the cellular immune response of a patient to removedisease-associated amyloid fibril deposits.
 11. The immunoglobulinpolypeptide or fragment thereof of claim 10, wherein the immunoglobulinpolypeptide or fragment thereof is a monoclonal antibody or fragmentthereof.
 12. The immunoglobulin or fragment thereof of claim 11, whereinthe monoclonal antibody is a humanized antibody.
 13. The immunoglobulinpolypeptide or fragment thereof of claim 11, wherein the monoclonalantibody is a chimeric antibody.
 14. The immunoglobulin polypeptide orfragment thereof of claim 13, wherein the chimeric antibody is ahumanized antibody.
 15. The immunoglobulin polypeptide or fragmentthereof of claim 11, wherein the antibody is a labeled antibody.
 16. Theimmunoglobulin polypeptide or fragment thereof of claim 11, wherein themonoclonal antibody is selected from the group consisting of κ1(57-18H12), κ4 (11-1F4), λ8 (31-8C7), and combinations thereof.
 17. Themonoclonal antibody or fragment thereof of claim 16, wherein themonoclonal antibody is a humanized antibody.
 18. The immunoglobulinpolypeptide or fragment thereof of claim 10, wherein the immunoglobulinpolypeptide or fragment thereof has been raised against syntheticamyloid fibrils.
 19. A pharmaceutical composition comprising theimmunoglobulin peptide or fragment thereof of claim
 10. 20. A nucleicacid molecule which encodes a polypeptide comprising at least ahypervariable region of the immunoglobulin polypeptide of claim
 10. 21.A host cell comprising a nucleic acid molecule of claim
 20. 22. A methodof producing an immunoglobulin polypeptide comprising the step ofculturing the host cell of claim 21.